Yalai Bai

L-Selectin-Dependent Lymphoid Occupancy Is Required to Induce Alloantigen-Specific Tolerance

Maneuvers that interfere with signals 1, 2, 3, or Ag processing can result in indefinite allograft survival. However, they are not applicable to all tissues, strains, or species, suggesting that there are additional levels of immune regulation. We hypothesized that secondary lymphoid organs are important for interactions among lymphocytes, alloantigen, and immunosuppressants that lead to tolerance. To explore this, cardiac allografts were performed with a tolerogenic immunosuppressive regimen. Concurrent administration of anti-L-selectin (CD62L) Ab, which prevents lymph node homing, prevents indefinite allograft survival and tolerance. Anti-CD62L Ab is not costimulatory, and Fab and F(ab′)2 anti-CD62L have similar activities. Flow cytometry and histologic examination show that Ab shifts T cells away from lymph nodes and into spleen, peripheral blood, and graft. Tolerance is not induced in CD62L−/− mice, and adoptive transfer of CD62L−/−, but not CD62L+/+, T cells prevents tolerization in wild-type recipients. FTY720, an immunosuppressant that promotes chemokine-dependent, but CD62L-independent, lymph node homing, reverses the Ab effect. Blockade of other homing receptors also prevents tolerization. These results indicate that T lymphocytes use CD62L-dependent migration for alloantigen-specific tolerance, and suggest that lymph nodes or other lymphoid tissues are an important site for peripheral tolerization to alloantigen.

Quantitative assessment shows loss of antigenic epitopes as a function of pre-analytic variables.

Pre-analytic variables, specifically cold ischemic time, have been implicated as key variables in the measurement of proteins by immunohistochemistry. To determine the significance and magnitude of antigenic loss due to pre-analytic variables, we have compared protein antigenicity in core needle biopsies, with essentially no cold ischemic time, with that in routinely processed tumor resection specimens. Two cohorts of matched core needle biopsies and tumor resections were collected with 20 matched pairs and 14 matched pairs, respectively. Both series were analyzed by quantitative immunofluorescence using the AQUA® method. Epitopes phospho-ERK, total ERK, phospho-AKT, total AKT, phospho-S6K1, total S6K1, estrogen receptor (ER), Ki67, cytokeratin and GAPDH were assessed. Detection levels for all phospho-epitopes were significantly decreased in tumor resections compared with biopsies while no significant change was seen in the corresponding total proteins. Of the other four proteins examined, ER and cytokeratin showed significant loss of antigenicity. This data suggest that measurement of phospho-protein antigenicity in formalin-fixed tissue by immunological methods is dramatically affected by pre-analytic variables. This study suggests that core needle biopsies are more accurate for assessment of tissue biomarkers.

Early and multiple origins of metastatic lineages within primary tumors

Significance The knowledge that cancer is an evolutionary process is old, but only recently can sequencing technology provide data for clinically relevant evolutionary analyses of cancer. Approaches developed for evolutionary biology can reveal the relationship among clonal lineages, the ancestral states of gene sequences, and the timing of evolutionary events. We performed whole exome sequencing of cancer tissues from multiple sites of dozens of subjects, demonstrating nonlinear patterns of tumor progression and early origins of metastatic lineages and quantifying the times of occurrence of driver mutations. These findings direct research attention away from the search for genes that induce metastasis toward genes that are mutated early in tumorigenesis, providing therapeutic targets effective against both primary tumors and metastases. Many aspects of the evolutionary process of tumorigenesis that are fundamental to cancer biology and targeted treatment have been challenging to reveal, such as the divergence times and genetic clonality of metastatic lineages. To address these challenges, we performed tumor phylogenetics using molecular evolutionary models, reconstructed ancestral states of somatic mutations, and inferred cancer chronograms to yield three conclusions. First, in contrast to a linear model of cancer progression, metastases can originate from divergent lineages within primary tumors. Evolved genetic changes in cancer lineages likely affect only the proclivity toward metastasis. Single genetic changes are unlikely to be necessary or sufficient for metastasis. Second, metastatic lineages can arise early in tumor development, sometimes long before diagnosis. The early genetic divergence of some metastatic lineages directs attention toward research on driver genes that are mutated early in cancer evolution. Last, the temporal order of occurrence of driver mutations can be inferred from phylogenetic analysis of cancer chronograms, guiding development of targeted therapeutics effective against primary tumors and metastases.

Combination of Electroporation and DNA/Dendrimer Complexes Enhances Gene Transfer into Murine Cardiac Transplants

Electroporation is a new gene delivery method to increase gene transfer and expression in vivo. Starburst polyamidoamine dendrimers have been demonstrated to augment gene expression in vitro and in vivo. We hypothesized that the combination of electroporation and dendrimer could enhance the gene transfer and gene expression in cardiac transplants. After immersion in DNA/dendrimer complexes or intracoronary transfer of DNA/dendrimer complexes, both nonvascularized and vascularized syngeneic cardiac grafts, respectively, were subjected to serial electrical pulses before transplantation. β‐Galactosidase reporter gene expression in the graft was determined by X‐Gal staining. Gene expression was enhanced 10‐ to 45‐fold in grafts immersed in DNA/dendrimer complexes, or after intracoronary transfer of DNA/dendrimer complexes, and subjected to 20 square wave 25‐ms pulses with a strength of 200 V/cm. The combination of electroporation and DNA/dendrimer complexes may provide a novel approach to enhance gene transfer and gene expression ex vivo.

A tissue quality index: an intrinsic control for measurement of effects of preanalytical variables on FFPE tissue

While efforts are made to improve tissue quality and control preanalytical variables, pathologists are often confronted with the challenge of molecular analysis of patient samples of unknown quality. Here we describe a first attempt to construct a tissue quality index (TQI) or an intrinsic control that would allow a global assessment of protein status based on quantitative measurement of a small number of selected, informative epitopes. Quantitative immunofluorescence (QIF) of a number of proteins was performed on a series of 93 breast cancer cases where levels of expression were assessed as a function of delayed time to formalin fixation. A TQI was constructed based on the combination of proteins that most accurately reflect increased and decreased levels of expression in proportion to delay time. The TQI, defined by combinations of measurements of cytokeratin, ERK1/2 and pHSP-27 and their relationship to cold ischemic time were validated on a second build of the training series and on two independent breast tissue cohorts with recorded time to formalin fixation. We show an association of negative TQI values (an indicator for loss of tissue quality) with increasing cold ischemic time on both validation cohorts and an association with loss of ER expression levels on all three breast cohorts. Using expression levels of three epitopes, we can begin to assess the likelihood of delayed time to fixation or decreased tissue quality. This TQI represents a proof of concept for the use of epitope expression to provide a mechanism for monitoring tissue quality.

Optimal tumor sampling for immunostaining of biomarkers in breast carcinoma

IntroductionBiomarkers, such as Estrogen Receptor, are used to determine therapy and prognosis in breast carcinoma. Immunostaining assays of biomarker expression have a high rate of inaccuracy; for example, estimates are as high as 20% for Estrogen Receptor. Biomarkers have been shown to be heterogeneously expressed in breast tumors and this heterogeneity may contribute to the inaccuracy of immunostaining assays. Currently, no evidence-based standards exist for the amount of tumor that must be sampled in order to correct for biomarker heterogeneity. The aim of this study was to determine the optimal number of 20X fields that are necessary to estimate a representative measurement of expression in a whole tissue section for selected biomarkers: ER, HER-2, AKT, ERK, S6K1, GAPDH, Cytokeratin, and MAP-Tau.MethodsTwo collections of whole tissue sections of breast carcinoma were immunostained for biomarkers. Expression was quantified using the Automated Quantitative Analysis (AQUA) method of quantitative immunofluorescence. Simulated sampling of various numbers of fields (ranging from one to thirty five) was performed for each marker. The optimal number was selected for each marker via resampling techniques and minimization of prediction error over an independent test set.ResultsThe optimal number of 20X fields varied by biomarker, ranging between three to fourteen fields. More heterogeneous markers, such as MAP-Tau protein, required a larger sample of 20X fields to produce representative measurement.ConclusionsThe optimal number of 20X fields that must be sampled to produce a representative measurement of biomarker expression varies by marker with more heterogeneous markers requiring a larger number. The clinical implication of these findings is that breast biopsies consisting of a small number of fields may be inadequate to represent whole tumor biomarker expression for many markers. Additionally, for biomarkers newly introduced into clinical use, especially if therapeutic response is dictated by level of expression, the optimal size of tissue sample must be determined on a marker-by-marker basis.

Comparison of HER2 and Phospho-HER2 Expression between Biopsy and Resected Breast Cancer Specimens Using a Quantitative Assessment Method

Background HER2/Neu (ErbB-2) overexpression, which occurs in 15–20% of breast cancer cases, is associated with better response to treatment with the drug trastuzumab. PhosphoHER2 (pHER2) has been evaluated for prediction of response to trastuzumab. Both markers are heterogeneously detected and are potentially subject to loss as a consequence of delayed time to fixation. Here, we quantitatively assess both markers in core needle biopsies (CNBs) and matched tumor resections to assess concordance between the core and the resection and between HER2 and pHER2. Methods A selected retrospective collection of archival breast cancer cases yielded 67 cases with both core and resection specimens. Both HER2 and pTyr1248HER2 were analyzed by the AQUA® method of quantitative immunofluorescence on each specimen pair. Results Both HER2 immunoreactivity (P<0.0001) and pTyr1248HER2 immunoreactivity (P<0.0001) were lower in resections relative to CNB specimens. However, clinical implications of this change may not be evident since no case changed from 3+ (CNB) to negative (resection). Assessment of pTyr1248HER2 showed no direct correlation with HER2 in either CNB or resection specimens. Conclusions The data suggest that measurement of both HER2 and phospho- Tyr1248HER2, in formalin-fixed tissue by immunological methods is significantly affected by pre-analytic variables. The current study warrants the adequate handling of resected specimens for the reproducible evaluation of HER2 and pHER2. The level of pTyr1248HER2, was not correlated to total HER2 protein. Further studies are required to determine the significance of these observations with respect to response to HER2 directed therapies.

CD2 is a Dominant Target for Allogeneic Responses

CD2 and 2B4 (CD244) are members of the immunoglobulin gene superfamily and are both ligands for another family member, CD48. CD2 is widely distributed on T, NK, and B cells and some antigen‐presenting cells, while 2B4 is expressed on NK and some T cells and monocytes and is known to participate in NK cytotoxicity. Since indefinite allograft survival could be obtained by a combination of anti‐CD48 plus anti‐CD2 mAb administration, it was important to determine the role of 2B4 blockade in allograft rejection. MAbs directed against CD2, CD48, or 2B4 were administered singly or in pairs to cardiac allograft recipients. The experiments show that only anti‐CD2 plus anti‐CD48 mAbs result in indefinite allograft survival, while anti‐CD2 plus anti‐2B4 mAbs substantially prolong graft survival, and anti‐CD48 plus anti‐2B4 mAbs were no better than each mAb alone. The effect of these mAbs on anti‐CD3 mAb and alloantigen‐driven proliferation and IFN‐γ production were also assessed. In general, anti‐CD2 inhibited both anti‐CD3 mAb and alloantigen‐driven responses, while anti‐CD48 inhibited only anti‐CD3 mAb but not alloantigen‐driven proliferative and cytokine responses. Anti‐2B4 mAbs were generally ineffective alone. Combinations of mAbs were more effective than single mAbs only in alloantigen‐driven proliferation, commensurate with allograft survival results. Using CD2–/– and CD48–/– T cells and antigen‐presenting cells, we also demonstrate that these inhibitory mAbs act primarily by blocking intercellular interactions, rather than directly delivering negative signals to T cells. These results suggest that, unlike CD2, 2B4 is not a potent regulatory molecule or ligand for CD48 in the response to alloantigen. Blocking the 2B4–CD48 receptor–ligand pair does not inhibit T‐cell responses and alloreactivity to the same degree as CD2–CD48 blockade.

Objective, domain-specific HER2 measurement in uterine and ovarian serous carcinomas and its clinical significance

Introduction HER2 overexpression/amplification is identified in up to 40% of uterine serous carcinomas (USC) and 10% of ovarian serous carcinomas (OSC). However, clinical trials using various HER2-targeted agents failed to show significant responses. FDA-approved HER2 assays target only the proteins intracellular domain (ICD) and not the extracellular domain (ECD). Previous quantitative studies in breast cancer by our group have shown that ICD of HER2 is expressed in some cases that do not express the HER2 ECD. We measured HER2 ICD and ECD in USC and OSC samples, and determined their relationship with clinico-pathologic characteristics and survival. Methods We measured HER2 ICD and ECD levels in 2 cohorts of USC and OSC comprising 102 and 175 patients, respectively. HER2 antibodies targeting ICD (CB11) and ECD (SP3) were validated and standardized using the AQUA® method of quantitative immunofluorescence (QIF) and a previously reported HER2 standardization tissue microarray (TMA). Objective, population-based cut-points were used to stratify patients according to HER2 ICD/ECD status. Results In USC, 8% of patients with high HER2 ICD had low ECD levels (6/75 patients). In OSC, 42% of patients with high HER2 ICD had low ECD levels (29/69 patients). HER2 ICD/ECD status in USC and OSC was not significantly associated with major clinico-pathological features or survival. Conclusion Using objective, domain-specific HER2 measurement, 8% of USC and 42% of OSC patients with high HER2 ICD levels do not show uniform overexpression of the ECD. This may be related to the presence of p95 HER2, an oncogenic fragment generated by full protein cleavage or alternative initiation of translation. These observations raise the possibility that USC/OSCs expressing low ECD despite being HER2-positive by ICD measurement, may benefit from therapies directed against the intracellular domain (e.g. lapatinib or afatinib) alone or in combination with extracellular domain-directed drugs (e.g. trastuzumab, pertuzumab, T-DM1).

Dual CCNE1/PIK3CA targeting is synergistic in CCNE1-amplified/PIK3CA-mutated uterine serous carcinomas in vitro and in vivo.

Background:Clinical options for patients harbouring advanced/recurrent uterine serous carcinoma (USC), an aggressive variant of endometrial tumour, are very limited. Next-generation sequencing (NGS) data recently demonstrated that cyclin E1 (CCNE1) gene amplification and pik3ca driver mutations are common in USC and may therefore represent ideal therapeutic targets.Methods:Cyclin E1 expression was evaluated by immunohistochemistry (IHC) on 95 USCs. The efficacy of the cyclin-dependent kinase 2/9 inhibitor CYC065 was assessed on multiple primary USC cell lines with or without CCNE1 amplification. Cell-cycle analyses and knockdown experiments were performed to assess CYC065 targeting specificity. Finally, the in vitro and in vivo activity of CYC065, Taselisib (a PIK3CA inhibitor) and their combinations was tested on USC xenografts derived from CCNE1-amplified/pik3ca-mutated USCs.Results:We found that 89.5% of the USCs expressed CCNE1. CYC065 blocked cells in the G1 phase of the cell cycle and inhibited cell growth specifically in CCNE1-overexpressing USCs. Cyclin E1 knockdown conferred increased resistance to CYC065, whereas CYC065 treatment of xenografts derived from CCNE1-amplified USCs significantly reduced tumour growth. The combination of CYC065 and Taselisib demonstrated synergistic effect in vitro and was significantly more effective than single-agent treatment in decreasing tumour growth in xenografts of CCNE1-amplified/pik3ca-mutated USCs.Conclusions:Dual CCNE1/PIK3CA blockade may represent a novel therapeutic option for USC patients harbouring recurrent CCNE1-amplified/pi3kca-mutated tumours.